Humidity control in chemical reactors

ABSTRACT

Control of humidity in chemical reactors, and associated systems and methods, are generally described. In certain embodiments, the humidity within gas transport conduits and chambers can be controlled to inhibit unwanted condensation within gas transport pathways. By inhibiting condensation within gas transport pathways, clogging of such pathways can be limited (or eliminated) such that transport of gas can be more easily and controllably achieved. In addition, strategies for purging condensed liquid from chemical reactor systems are also described.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/654,808, filed Oct. 16, 2019, which is a divisional of U.S. patentapplication Ser. No. 14/064,021, filed Oct. 25, 2013 (now U.S. Pat. No.10,472,602), which claims priority to U.S. Provisional PatentApplication Ser. No. 61/719,085, filed Oct. 26, 2012, and entitled“Humidity Control in Chemical Reactors”; U.S. Provisional PatentApplication Ser. No. 61/869,118 filed Aug. 23, 2013, and entitled“Humidity Control in Chemical Reactors”; and European Application No.13306464.2 filed Oct. 23, 2013 and entitled “Humidity Control inChemical Reactors,” each of which is incorporated herein by reference inits entirety for all purposes.

TECHNICAL FIELD

Systems and methods for the control of humidity in chemical reactors aregenerally described.

BACKGROUND

There is currently a great deal of interest in developing small volumebioreactors for growing cells, for example, for biopharmaceuticalproduction. Controlling liquid levels in such reactors can bechallenging. For example, evaporation of small amounts of liquid mediumwithin the reactor can lead to relatively large changes in volume, whichcan adversely impact bioreactor operation. Improved systems and methodsfor controlling humidity in such chemical reactors are thereforedesirable.

SUMMARY

Control of humidity in chemical reactors, as well as associated systemsand methods, are generally described. Certain embodiments relate to thecontrol of humidity within gas transport conduits. The subject matter ofthe present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

In certain embodiments, a reactor system is provided. In someembodiments, the reactor system comprises a reactor chamber; a reactorchamber gas inlet conduit configured to transport gas into the reactorchamber through a reactor chamber gas inlet; a flow control mechanismconfigured to regulate the flow of gas through the reactor chamber gasinlet conduit at a rate of equal to or less than about 1 milliliter persecond; and a humidifier configured to humidify the gas transportedthrough the reactor chamber gas inlet conduit, the humidifier positionedbetween the flow control mechanism and the reactor chamber gas inlet.

In some embodiments, the reactor system comprises a reactor chamber; areactor chamber gas outlet conduit configured to transport gas out ofthe reactor chamber through a reactor chamber gas outlet; a flow controlmechanism configured to regulate the flow of gas through the reactorchamber gas outlet conduit at a rate of equal to or less than about 1milliliter per second; and a liquid trap configured to remove liquidvapor from the gas within the reactor chamber gas outlet conduit, theliquid trap positioned between the flow control mechanism and thereactor chamber gas outlet.

The reactor system comprises, in certain embodiments, a reactor chamber,comprising a liquid sub-chamber configured to contain a liquid growthmedium including at least one biological cell; a gas sub-chamberconfigured to contain a gaseous headspace above the liquid growthmedium; and a gas-permeable flexible membrane separating the liquidsub-chamber from the gas sub-chamber. In certain embodiments, thereactor system comprises a gas inlet conduit configured to transport gasinto the gas sub-chamber; a gas outlet conduit configured to transportgas out of the gas sub-chamber; and a gas bypass conduit external to thereactor chamber, connecting the gas inlet conduit to the gas outletconduit.

Certain embodiments relate to methods of operating a reactor. In certainembodiments, the method comprises providing a reactor chamber,comprising a liquid sub-chamber configured to contain a liquid growthmedium including at least one biological cell; a gas sub-chamberconfigured to contain a gaseous headspace above the liquid growthmedium; and a flexible membrane separating the liquid sub-chamber fromthe gas sub-chamber. In some embodiments, the method comprisestransporting a gas from a gas source through a gas inlet conduit to thegas sub-chamber to deform the gas-permeable flexible membrane such thatliquid is at least partially removed from the liquid sub-chamber;reducing the supply of the gas to the gas sub-chamber such that theflexible membrane returns toward its original position; transporting gasfrom the gas source through a gas bypass connected to the gas inletconduit and external to the reactor chamber to remove liquid from thegas inlet conduit; and supplying gas from the gas source to the gassub-chamber at least a second time to deform the gas-permeable flexiblemembrane such that liquid is at least partially evacuated from theliquid sub-chamber.

In certain of the above embodiments, the biological cell is a eukaryoticcell.

In some of the above embodiments, the reactor chamber is configured tocontain a volume of the liquid medium that is equal to or less thanabout 50 milliliters and equal to or greater than 10 microliters.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a cross-sectional schematic illustration of a reactor system,according to certain embodiments;

FIGS. 2A-2C are cross-sectional schematic illustrations of a reactorchamber and a mode of operating the same, according to one set ofembodiments;

FIG. 3 is a bottom-view cross sectional schematic illustration of areactor system including a plurality of reactor chambers arranged inseries;

FIG. 4A-B are A) a cross-sectional schematic illustration of a reactorsystem with a long path and B) a cross-sectional schematic illustrationof a reactor system with a short path, according to certain embodiments;

FIG. 5 is a cross-sectional schematic illustration of a reactor system,according to certain embodiments;

FIG. 6 is a cross-sectional schematic illustration of a gas manifold fora reactor system, according to one set of embodiments;

FIG. 7 is a cross-sectional schematic illustration of a gas manifold fora reactor system, according to some embodiments; and

FIG. 8 is a photograph of a reactor system, according to certainembodiments.

DETAILED DESCRIPTION

Control of humidity in chemical reactors, and associated systems andmethods, are generally described. In certain embodiments, the humiditywithin gas transport conduits and chambers can be controlled to inhibitunwanted condensation within gas transport pathways. By inhibitingcondensation within gas transport pathways, clogging of such pathwayscan be limited (or eliminated) such that transport of gas can be moreeasily and controllably achieved. In addition, strategies for purgingcondensed liquid from chemical reactor systems are also described.

The embodiments described herein can be used to control evaporation andcompensate for liquid loss in reactor chambers. Such control andcompensation can be especially useful in small volume reactors (e.g.,reactors having volumes of about 50 milliliters or less), in which theloss of even small amounts of liquids can adversely impact reactorperformance. In certain embodiments, the reactors described hereininclude a liquid phase (which can contain, for example, a liquid growthmedium for biological cells such as any common cell growth mediumcontaining essential amino acids and cofactors known to those ofordinary skill in the art) and a gas phase (e.g., comprising carbondioxide, oxygen, and/or an inert gas). In some such embodiments, theliquid phase and the gas phase can be in direct contact, while in othersuch embodiments, the liquid phase and the gas phase can be separated bya moveable wall, as described in more detail below.

In certain embodiments, a humidifier can be connected directly orindirectly to a gas inlet of the reactor chamber. The humidifier can beany vessel in which gas is transported (e.g., bubbled) through a liquidat a temperature that is equal to or higher (e.g., at least about 1° C.higher, at least about 5° C. higher, at least about 10° C. higher, atleast about 20° C. higher, at least about 30° C. higher, or at leastabout 40° C. higher (and/or, in certain embodiments, up to about 50° C.higher, up to about 75° C. higher, or more)) than the temperature of theliquid in the reactor chamber (e.g., between about 30° C. to about 40°C.). The humidifier can be configured to produce a humidifier gas outletstream having a liquid vapor content that is greater than the liquidvapor content of the gas transported into the humidifier. For instance,in some embodiments, the liquid vapor content is greater than or equalto about 70% (e.g., greater than or equal to about 80%, greater than orequal to about 90%, about 100%) of the saturation point for thehumidifier temperature.

In some embodiments, a liquid trap can be connected to a gas outlet ofthe reactor chamber. The liquid trap can be any vessel in which gas istransported (e.g., bubbled) through a liquid at a temperature that isequal to or lower (e.g., at least about 1° C. lower, at least about 5°C. lower, least about 10° C. lower, or least about 20° C. lower) thanthe temperature of the liquid in the reactor chamber. The liquid trapcan be configured to produce a liquid trap gas outlet stream having aliquid vapor content that is lower than the liquid vapor content of thegas transported into the liquid trap. For example, the liquid vaporcontent of the gas may be from about 0% to about 10% (e.g., from about0% to about 5%).

In certain embodiments, liquid can be removed from gas transport linesleading to one or more inlet(s) and/or outlet(s) of the reactor chamber,for example, by flushing the lines with gas during a period of time inwhich there is one or more unblocked connection between an inlet andoutlet of the chamber, either designed into the chamber or temporarilyunblocked due to a state of the chamber.

In certain embodiments, the gas flow rate through the reactor chambercan be reduced (e.g., by at least about 80%, by at least about 90%, byat least about 95% (and/or, in some embodiments, by up to about 99%, ormore)) by using a constriction (or other flow rate regulation device) inthe gas transport conduits leading into and/or out of the chamber. Insome such embodiments in which a humidifier is present, the humidifiercan be located between the inlet constriction and the inlet of thereactor chamber. In some such embodiments in which a liquid trap ispresent, the liquid trap can be located between the reactor chamber andthe outlet constriction. Such positioning of the constriction can ensurethat clogging of the constrictions with liquid is inhibited oreliminated.

FIG. 1 is a schematic illustration of a reactor system 100. Reactorsystem 100 can comprise a reactor chamber 102. Reactor system 100 canfurther comprise a reactor chamber gas inlet conduit 104. Reactorchamber gas inlet conduit 104 can be configured to transport gas intoreactor chamber 102 through reactor chamber gas inlet 106.

Reactor system 100 can also comprise a flow control mechanism 108configured to regulate the flow of gas through the reactor chamber gasinlet conduit. A variety of suitable devices can be used as flow controlmechanisms. For example, in certain embodiments, including thoseillustrated in FIG. 1 , flow control mechanism 108 corresponds to aconstriction in a gas supply conduit. The cross-sectional dimension ofthe gas supply conduit within the constriction can be, in certainembodiments, at least about 10% smaller, at least about 25% smaller, atleast about 50% smaller, at least about 80% smaller, at least about 90%smaller, or at least about 95% smaller (and/or, in some embodiments, upto about 99% smaller, or smaller) than the smaller of the upstream anddownstream cross-sectional diameters of the gas supply conduit, incertain embodiments. In other embodiments, flow control mechanism 108corresponds to a pressure regulator, which generally automatically cutsoff the flow of a gas at a certain pressure. In general, any suitableflow control mechanism that poses a resistance to flow may be used, suchas, for example, constrictions, extensions of the gas supply conduit,impedance material in the gas supply conduit (e.g., filters), and thelike.

In certain embodiments, flow control mechanism 108 is configured toregulate the flow of gas through the reactor chamber gas inlet conduitat a relatively low flow rate. For example, in certain embodiments, flowcontrol mechanism 108 is configured to regulate the flow of gas throughthe reactor chamber gas inlet conduit at a flow rate of equal to or lessthan about 1 milliliter per second, equal to or less than about 100microliters per second, or equal to or less than about 10 microlitersper second (and/or in certain embodiments, as low as about 0.1microliters per second). Relatively slow transport of gas through areactor system can be important, for example, in small-scale reactors,which might require relatively slow flows of gas to the reactor chamber.

In certain embodiments, reactor system 100 comprises a humidifier 110.Humidifier 110 can be configured to humidify the gas transported throughreactor chamber gas inlet conduit 104. The humidifier can comprise, forexample, a fluid contained within a vessel. The gas inlet conduit to thehumidifier can have an outlet that is submerged in the fluid such thatthe gas is bubbled through the fluid within the humidifier.Subsequently, the gas can be transported out of the humidifier via a gasoutlet conduit. One of ordinary skill in the art would be capable ofdesigning other schemes to achieve humidification of the gas transportedthrough reaction chamber gas inlet conduit 104.

In certain embodiments, humidifier 110 is positioned between flowcontrol mechanism 108 and reactor chamber gas inlet 106. That is to say,humidifier 110 can be fluidically connected such that, after gas istransported out of flow control mechanism 108, the gas is subsequentlytransported through humidifier 110, and subsequently to reactor chamber102. Positioning the humidifier in this way can reduce the degree towhich liquid condenses within reactor chamber gas inlet conduit 104. Incases in which humidifier 110 is placed upstream of flow controlmechanism 108, the relatively humid gas exiting the humidifier can bemore prone to condense while moving relatively slowly through the narrowpassageways of flow control mechanism 108. On the other hand, whenhumidifier 110 is placed downstream of flow control mechanism 108, thegas that is transported through flow control mechanism 108 is relativelydry, and condensation can be inhibited (or eliminated).

Humidifier 110 can be used, for example, to supply liquid to or maintainthe level of liquid within reactor chamber 102. For example, if theliquid within reactor chamber 102 evaporates or is otherwise removedfrom the reaction chamber during operation, the liquid within the gassupplied by reactor chamber gas inlet conduit 104 can be transferredfrom the gas within reactor chamber 102 to the liquid within reactionchamber 102 (optionally, through a moveable wall such as a membrane,discussed in more detail below). If the liquid level within reactorchamber 102 is determined to be at a desired level, the amount of liquidin the gas supplied by reactor chamber gas inlet conduit 104 can be set(e.g., using humidifier 110) such that evaporation of liquid withinreactor chamber 102 is inhibited or eliminated.

In some embodiments, reactor system 100 includes a reactor chamber gasoutlet conduit 112. Reactor chamber gas outlet conduit 112 can beconfigured to transport gas out of the reactor chamber through a reactorchamber gas outlet 114. For example, gas may be transported out of thereactor chamber after a moveable wall (e.g., a flexible membrane) hasbeen actuated, as discussed in more detail below with respect to FIGS.2A-2C and FIG. 3 . Gas might also be transported out of the reactorchamber after oxygen and/or CO₂ within the gas has been transported fromthe gaseous headspace to the liquid medium within the reactor chamber.

In certain embodiments, reactor system 100 includes a flow controlmechanism 116 configured to regulate the flow of gas through reactorchamber gas outlet conduit 112. Any suitable device can be used in flowcontrol mechanism 116, including any of those outlined above withrespect to flow control mechanism 108. In certain embodiments, flowcontrol mechanism 116 is configured to regulate the flow of gas throughthe reactor chamber gas inlet conduit at a relatively low flow rate(e.g., at a rate of equal to or less than about 1 milliliter per second,or at any other rate mentioned above with respect to flow controlmechanism 108).

Reactor system 100 comprises, in certain embodiments, liquid trap 118.Liquid trap 118 can be configured to remove liquid vapor from the gaswithin reactor chamber gas outlet conduit 112. Liquid trap 118 can beused, for example, to measure the amount of liquid exiting reactorchamber 102 (e.g., by measuring the change in the amount of liquidcontained in the liquid trap). By determining the amount of liquidexiting reactor chamber 102, one can determine whether liquid is beinglost from the reactor chamber without directly measuring the amount ofliquid within the reactor chamber (which can be difficult to do, incertain circumstances).

In some embodiments, liquid trap 118 is positioned between flow controlmechanism 116 and reactor chamber gas outlet 114. That is to say, liquidtrap 118 can be fluidically connected such that, after gas istransported out of reactor chamber 102, the gas is subsequentlytransported through liquid trap 118, and subsequently to flow controlmechanism 116. Positioning the liquid trap in this way can reduce thedegree to which liquid condenses within reactor chamber gas outletconduit 112. In cases in which liquid trap 118 is placed downstream offlow control mechanism 116, the relatively humid gas exiting the reactorchamber can be more prone to condense while moving relatively slowlythrough the narrow passageways of flow control mechanism 116. On theother hand, when liquid trap 118 is placed upstream of flow controlmechanism 116, the liquid trap can be used to remove vapor from the gasbefore it is transported to flow control mechanism 116 (such that thegas that is transported through flow control mechanism 116 is relativelydry), and condensation can be inhibited (or eliminated).

In certain embodiments, an amount of liquid added to or lost from thereactor chamber can be determined using the humidifier and/or the liquidtrap. This can be achieved, for example, by weighing the humidifier, thereactor chamber, and/or liquid trap. For example, one could measure theweight of the liquid in the humidifier, measure the weight of the liquidin the liquid trap, determine the liquid vapor content of the gas streamentering the humidifier, and determine the liquid vapor content of thegas stream exiting the liquid trap. After making such a determination, amass balance could be performed to determine the amount of liquid addedto or lost from the reactor chamber, optionally without weight thereactor chamber itself. As one illustrative example, if the amount ofliquid vapor in the gas stream entering the humidifier is equal to theamount of liquid vapor in the gas stream exiting the liquid trap, thenthe amount of liquid added to or lost from the reactor chamber can bedetermined by subtracting the change in weight of the liquid trap fromthe change in weight of the humidifier.

In some embodiments, the humidification and/or evaporation rate of theliquid in the reactor chamber can be determined by measuring thehumidity of the gas passing through the inlet and outlet of the reactorchamber.

In some embodiments, the uptake and/or release rate of a single orplurality of different gasses into the reactor chamber can be determinedby measuring the concentration of a single or plurality of gasses in theinlet and outlet of the chamber.

In certain embodiments, two or more (or all) of the strategies outlinedabove can be used in combination with each other.

While FIG. 1 illustrates a system in which both humidifier 110 andliquid trap 118 are present, certain embodiments may use only thehumidifier or only the liquid trap. For example, in certain embodiments,reactor system 100 includes humidifier 110, but does not include liquidtrap 118. In some embodiments, reactor system 100 includes liquid trap118, but does not include humidifier 110.

In certain embodiments, reactor chamber 102 comprises a gaseousheadspace and a liquid medium that are in direct contact. In otherembodiments, however, the gaseous headspace and liquid medium areseparated by a moveable wall. Reactors employing such arrangements aredescribed, for example, in U.S. patent application Ser. No. 13/249,959by Ram et al, filed Sep. 30, 2011, and entitled “Device and Method forContinuous Cell Culture and Other Reactions” and U.S. Patent ApplicationPublication No. 2005/0106045 by Lee, filed Nov. 18, 2003, and entitled“Peristaltic Mixing and Oxygenation System,” each of which isincorporated herein by reference in its entirety for all purposes.

FIGS. 2A-2C are cross-sectional schematic illustrations outlining howfluid can be transported by deflecting a moveable wall into and out of aliquid sub-chamber of a reactor chamber. In FIGS. 2A-2C, reactor system200 comprises reactor chamber 202. In certain embodiments, reactorchamber 202 in FIGS. 2A-2C corresponds to reactor chamber 102 in FIG. 1. Reactor chamber 202 can comprise a liquid sub-chamber 204. Liquidsub-chamber 204 can be configured to contain a liquid growth mediumincluding at least one biological cell. Reactor chamber 202 cancomprise, in certain embodiments, gas sub-chamber 206. Gas sub-chamber206 can be configured to contain a gaseous headspace above the liquidgrowth medium within liquid sub-chamber 204.

Reactor chamber 202 can also comprise a moveable wall 208, which canseparate liquid sub-chamber 204 from gas sub-chamber 206. Moveable wall208 can comprise, for example, a flexible membrane. In certainembodiments, the moveable wall is formed of a medium that is permeableto at least one gas (i.e., a gas-permeable medium). In certainembodiments, for example, moveable wall can be permeable to oxygen gasand/or carbon dioxide gas. In such embodiments in which moveable wall208 is permeable to a gas (e.g., oxygen and/or carbon dioxide), the gaswithin gas sub-chamber 206 can be transported to liquid sub-chamber 204,or vice versa. Such transport can be useful, for example, to transportoxygen gas into a liquid medium within liquid sub-chamber 204 and/orcontrol pH by transporting carbon dioxide into or out of liquidsub-chamber 204.

Reactor system 200 can comprise, in certain embodiments, a gas inletconduit 104, which can be configured to transport gas into gassub-chamber 206. Gas inlet conduit 104 in FIGS. 2A-2C can correspond tothe gas inlet conduit 104 illustrated in FIG. 1 , in certainembodiments. The gas that is transported into gas sub-chamber 206 canoriginate from, for example, gas source 216. Any suitable source of gascan be used as gas source 216, such as gas cylinders. In certainembodiments, gas source 216 is a source of oxygen and/or carbon dioxide.

In some embodiments, reactor system 200 comprises gas outlet conduit 112configured to transport gas out of gas sub-chamber 206. Gas outletconduit 112 in FIGS. 2A-2C can correspond to the gas outlet conduit 112illustrated in FIG. 1 , in certain embodiments. In some embodiments,reactor system 200 comprises gas bypass conduit 210 connecting gas inletconduit 104 to gas outlet conduit 112. Gas bypass conduit 210 can beconfigured such that it is external to reactor chamber 202, in certainembodiments. The set of embodiments illustrated in FIG. 1 can alsoinclude a gas bypass conduit, illustrated as conduit 210. Reactor system200 can also comprise, in certain embodiments, a liquid inlet conduit212 and a liquid outlet conduit 214.

In certain embodiments, moveable wall 208 can be actuated such that thevolumes of liquid sub-chamber 204 and gas sub-chamber 206 are modified.For example, certain embodiments involve transporting a gas from gassource 216 through gas inlet conduit 104 to gas sub-chamber 206 todeform moveable wall 208. Deformation of moveable wall 208 can beachieved, for example, by configuring reactor 200 such that gassub-chamber 206 is pressurized when gas is transported into gassub-chamber 206. Such pressurization can be achieved, for example, byrestricting the flow of gas out of gas outlet conduit 112 (e.g., usingvalves or other appropriate flow restriction mechanisms) while gas isbeing supplied to gas sub-chamber 206.

In certain embodiments, deforming moveable wall 208 can result in liquidbeing at least partially removed from liquid sub-chamber 204. Forexample, in FIG. 2B, moveable wall 208 has been deformed such thatsubstantially all of the liquid within liquid sub-chamber 204 has beenremoved from reactor chamber 202. Such operation can be used totransport the liquid within liquid sub-chamber 204 to other liquidsub-chambers in other reactors, as illustrated, for example, in FIG. 3 ,described in more detail below.

In certain embodiments, after at least a portion of the liquid withinliquid sub-chamber 204 has been removed from liquid sub-chamber 204, thesupply of the gas to gas sub-chamber 206 can be reduced such thatmoveable wall 208 returns toward its original position (e.g., theposition illustrated in FIG. 2A). In certain embodiments, moveable wall208 will be deflected such that at least a portion of the gas within gassub-chamber 206 is removed from the gas sub-chamber. Such gas might beremoved, for example, if liquid enters liquid sub-chamber 204 fromliquid inlet 212, for example, from another upstream reactor, asdescribed in more detail below.

Certain embodiments include the step of supplying gas from gas source216 to gas sub-chamber 206 at least a second time (and, in certainembodiments, at least 10 times, at least 100 times, or more) to deformmoveable wall 208 such that liquid is at least partially removed fromliquid sub-chamber 204. When such gas introduction steps are performedrepeatedly, moveable wall 208 can act as part of a pumping mechanism,transporting liquid into and out of liquid sub-chamber 204. Suchoperation is described in detail in U.S. patent application Ser. No.13/249,959 by Ram et al, filed Sep. 30, 2011, and entitled “Device andMethod for Continuous Cell Culture and Other Reactions.” In certainembodiments, the multiple steps of supplying gas from gas source 216 togas sub-chamber 206 can be performed relatively rapidly (e.g., incertain embodiments, at frequencies of between about 0.1 Hertz and about1000 Hertz, between about 0.5 Hertz and about 10 Hertz, or between about1 Hertz and about 3 Hertz).

In certain embodiments in which gas is transported into gas sub-chamber206 multiple times, gas can be transported from the gas source throughgas bypass conduit 210. Transporting gas through gas bypass conduit 210can be performed to remove liquid from gas inlet conduit 104 withouttransporting the liquid to gas sub-chamber 206. For example, in certainembodiments, a first valve between gas bypass conduit 210 and gas inlet106 can be closed and a second valve between gas bypass conduit 210 andgas outlet 114 can be closed (and any valves within gas bypass conduit210 can be opened) such that, when gas is transported through gas inletconduit 104, the gas is re-routed through gas bypass conduit 210, andsubsequently out gas outlet conduit 112. Such operation can serve toflush any unwanted condensed liquid out of the gas inlet conduit, whichcan improve the performance of the gas supply methods describedelsewhere herein. In certain embodiments, gas can be transported throughthe bypass conduit 210 (e.g., as described above) in between steps oftransporting gas into gas sub-chamber 206 (e.g., to actuate the moveablewall, as described below), which steps can be, as described above,performed relatively rapidly.

In some embodiments, multiple sets of reactor chambers can be arranged(e.g., in series) such that fluidic mixing is achieved along one or morefluidic pathways. FIG. 3 is a bottom view, cross-sectional schematicdiagram illustrating the liquid flow paths that can be used to establishmixing between multiple reactor chambers 102A-C connected in series, asdescribed in U.S. patent application Ser. No. 13/249,959 by Ram et al,filed Sep. 30, 2011, and entitled “Device and Method for Continuous CellCulture and Other Reactions.”

In FIG. 3 , reactor system 300 includes a first fluidic pathwayindicated by arrows 310. The first fluidic pathway can include a firstreactor chamber 102A, a second reactor chamber 102B, and a third reactorchamber 102C. Reactor system 300 also includes conduits 321, 322, and323, which can correspond to liquid inlet and/or liquid outlet conduitsfor reactor chambers 102A-C. For example, in FIG. 3 , conduit 321 is aliquid inlet conduit for reactor chamber 102B and a liquid outletconduit for reactor chamber 102A; conduit 322 is a liquid inlet conduitfor reactor chamber 102C and a liquid outlet conduit for reactor chamber102B; and conduit 323 is a liquid inlet conduit for reactor chamber 102Aand a liquid outlet conduit for reactor chamber 102C. Of course, theflow of liquid can also be reversed such that conduits 321, 322, and 323assume opposite roles with respect to each of reactor chambers 102A-C.

Reactor system 300 can also include a liquid input conduit 350 and aliquid output conduit 351, which can be used to transport liquid intoand out of the liquid sub-chambers within reactor chambers 102A, 102B,and 102C. Valve 352 may be located in liquid input conduit 350, andvalve 353 may be located in liquid output conduit 351 to inhibit orprevent to the flow of liquid out of the mixing system during operation.

In certain embodiments, the moveable walls of reactor chambers 102A-Ccan be actuated to transport liquid along fluidic pathway 310 (and/oralong a fluidic pathway in a direction opposite pathway 310). This canbe achieved, for example, by sequentially actuating the moveable wallswithin reactor chambers 102A-C such that liquid is transported in acontrolled direction. In some embodiments, each of reactor chambers102A-C can be configured such that they are each able to assume a closedposition wherein moveable wall 208 is strained such that the volume ofthe liquid sub-chamber is reduced, for example, as illustrated in FIG.2B. Peristaltic mixing can be achieved, for example, by actuatingreactor chambers 102A-C such that their operating states alternatebetween open (FIGS. 2A or FIG. 2C) and closed (FIG. 2B) configurations.In some embodiments, three patterns may be employed to achieveperistaltic pumping: a first pattern in which the liquid sub-chamber ofreactor chamber 102A is closed and the liquid sub-chambers withinreactor chambers 102B and 102C are open; a second pattern in which theliquid sub-chamber of reactor chamber 102B is closed and the liquidsub-chambers within reactor chambers 102A and 102C are open; and a thirdpattern in which the liquid sub-chamber of reactor chamber 102C isclosed and the liquid sub-chambers within reactor chambers 102A and 102Bare open. By transitioning among these three patterns (e.g., changingfrom the first pattern to the second pattern, from the second pattern tothe third pattern, and from the third pattern to the first pattern,etc.) liquid can be transported among reactor chambers 102A-C in aclockwise direction (as illustrated in FIGS. 2A-2B). Of course, byre-arranging the order in which the patterns occur (e.g., by changingfrom the first pattern to the third pattern, from the third pattern tothe second pattern, and from the second pattern to the first pattern,etc.), liquid can be transported in the counter-clockwise direction aswell.

In certain embodiments, the reactor systems described herein can be usedas bioreactors. For example, the reactor systems can be configured toculture biological cells. In some such embodiments, a liquid growthmedium containing at least one cell is contained within the reactorchamber to achieve cell growth. The liquid growth medium can contain anytype of biological cell or cell type. For example, the cell may be abacterium (e.g., E. coli) or other single-cell organism, a plant cell,or an animal cell. In some embodiments, the cell may be a eukaryoticcell. If the cell is a single-cell organism, then the cell may be, forexample, a protozoan, a trypanosome, an amoeba, a yeast cell, algae,etc. If the cell is an animal cell, the cell may be, for example, aninvertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g., azebrafish cell), an amphibian cell (e.g., a frog cell), a reptile cell,a bird cell, or a mammalian cell such as a primate cell, a bovine cell,a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell, or acell from a rodent such as a rat or a mouse. In some embodiments, thecell can be a human cell. In some embodiments, the cell may be a hamstercell, such as a Chinese hamster ovary (CHO) cell. If the cell is from amulticellular organism, the cell may be from any part of the organism.For instance, if the cell is from an animal, the cell may be a cardiaccell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, aneural cell, a osteocyte, a muscle cell, a blood cell, an endothelialcell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, aneutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.In some cases, the cell may be a genetically engineered cell.

The reactor chamber can, in some embodiments, be configured to contain(and/or, can contain during operation of the reactor) a volume of liquidmedium equal to or less than about 50 milliliters, equal to or less thanabout 10 milliliters, or equal to or less than about 2 milliliters(and/or, in certain embodiments, equal to or greater than 10microliters, equal to or greater than 100 microliters, or equal to orgreater than 1 milliliter). In certain embodiments, the reactor chamberhas an aspect ratio of less than about 10 (or less than about 8, such asbetween about 5 and about 8), as measured by dividing the largest crosssectional dimension of the chamber by the smallest cross-sectionaldimension of the chamber.

The following example is intended to illustrate certain embodiments ofthe present invention, but does not exemplify the full scope of theinvention.

Example

This example describes the design and operation of a reactor systemintegrating inventive humidity control methods.

In many bioreactor systems, problems surface when the micro-bioreactoris used for long-term cultures (10-14 days). For instance, for long termcultures, evaporation becomes a much larger problem. Evaporation cancause relatively small reactors to lose 20% of the working liquid volumewithin the reactor per day, or more. Even after gas inlet lines arehumidified, evaporation of liquid medium out of the reactor can still bea problem. Theoretically, if the air above the liquid medium in thereactor is humidified and completely saturated at 37° C., there shouldbe no evaporation since the air above is saturated with water vapor forthe given pressure and temperature. However, even if air is saturated at80° C. upon entering a gas inlet line, the air might not be saturatedany longer once it reaches the reactor chamber if the gas transportconduit is sufficiently long.

Even when humidification it is often necessary to take measures toinhibit liquid loss and prevent the culture from going into highosmolarity. For example, evaporation compensation by injecting sterilewater can be used for long term cultures. This can be achieved, forexample, by refilling the reactor chamber with sterile water (e.g.,every few hours). Such methods can work well for fixed working volumecultures, such as chemostat cultures. However, for many other cultures,including certain CHO cultures, the volume of the culture medium mightbe irregular throughout the culture due to offline sampling and feeding.In such cultures, it can be very challenging (or impossible) toimplement closed loop correction for evaporation at all times, andgenerally, the evaporation rate would need to be determined beforehandthrough a separate experiment. Other methods of evaporation compensationare known, but most require the volume of the micro-bioreactor to bekept constant throughout the culture.

On possible alternative method for closed loop evaporation compensationwithout requiring the working volume to be kept constant would be toconstantly weigh the local humidifier and the water trap and compensatefor any weight increase by injecting the equivalent amount of water intothe growth chamber.

In many CHO cultures, the slow mixing rate for culturing the CHO cellscan cause water to condense within the air lines, a problem that is notseen as much when air flow is faster, as might be observed in manybacteria cultures. Condensation can be especially problematic whenremote humidifiers are used as shown in FIG. 4A due to the long paththat is unheated between the humidifier and the micro-bioreactor. Theproblem can be further exacerbated when there are air resistance lines,such as air resistance lines configured to reduce shear stress in thegrowth chamber. The viscosity of water is two orders of magnitude higherthan air and in the narrow resistance channels. Accordingly, when waterplugs the air channels, the flow rate of water is so slow that themembrane does not deflect at all within the duration of the mixeractivation. Moreover, it is believed that more condensation occurs withthe presence of stainless steel parts (e.g., which might be present in asolenoid valve within the gas line) are in contact with humidified airdue the higher thermal conductivity of metal. Water condensation on thesolenoid valves, besides reducing the effectiveness of humidification,also can cause failures of solenoid valves over time.

A new humidification strategy is illustrated in the schematicillustration of FIG. 4B. The new strategy employs a local humidifier(e.g., set at 45° C.) and a water trap (e.g., set at 25° C.). Thisconfiguration prevents the humidified air from ever coming into contactwith the solenoid valves, which will ensure a longer lifetime of thevalves. Also, in this configuration, the resistance lines are placedbefore the humidifier and after the water trap; accordingly, only dryair passes through the resistance lines to prevent any potentialclogging of the resistance lines with condensed water. While the air inthe humidifier will equilibrate for a shorter period of time (relativeto the amount of time it would have to equilibrate when placed at thebeginning of the air lines), it is believed that direct water injectionsinto the growth chamber will be sufficient to compensate for the lostliquid. Also, the additional water trap can act as a hydraulicequivalent of a capacitor on the mixer resistance, allowing the membranedeflection time to be increased since a high capacitance can increasethe time constant of the deflection. This means that the resistancechannels can be made wider and if there is any residual water in thechannel, the droplet will be able to move faster in a wider channel.Moreover, the mixer now has separate input and output gas lines toenable flushing of the lines in case any part of the air lines getclogged. This flushing process will be performed periodically throughthe experiments and runs through the bypass conduit on top of themixers, as shown in FIG. 4B.

In order to perform an open loop evaporation compensation, theevaporation rate for the RECA Micro-bioreactor should be characterizedprior to performing the cell culture experiment. The increase inconcentration of green food dye injected into the growth chamber is usedas a parameter to calculate the evaporation rate. For this measurement,an intensive variable measurement will be more accurate than anextensive variable (e.g. volume or mass) measurement since the error islarger for an extensive variable and the characterization will beheavily dependent on the experimental procedure. Since the RECAMicro-bioreactor has an optical density (OD) sensor integrated, usingthe OD sensor to measure the light scattered/absorbed from the green dyeis an accurate way to obtain the evaporation rate, α. The increase indye concentration as a function of time will follow the followingrelation:

${C(t)} = \frac{C_{o}}{1 - {( {\alpha/V} )t}}$where C(t) is the concentration of the food dye as a function of time,C_(o) is the initial concentration of food dye and V is the volume ofliquid in the growth chamber (2 mL). Since the evaporation rate is notexpected to be very high, typically of the order of 1-10 μL/hr, theexperiment is performed overnight (7-8 hours) to obtain a higheraccuracy. The measured evaporation rate with a local 45° C. humidifierattached is 4.7 μL/hr. If this evaporation is uncompensated, 75% of thevolume of the micro-bioreactor will have evaporated by the end of a 14day experiment. This could significantly increase the osmolarity of themedium and inhibit growth.

The strategies outlined above can be used with a new reactor design,referred to in this example as the Resistive Evaporation CompensatedActuator (RECA) micro-bioreactor, which is illustrated in FIG. 5 . Thereactor includes 5 reservoirs for injections, including one containingsterile water for evaporation compensation. The other four reservoirscan be used for Sodium Bicarbonate (NaHCO₃) base injections, feed, andother necessary supplements. Injection can be performed by a peristalticpump actuated through the PDMS membrane sequentially pushing a plug offluid into the growth chamber. In this example, the growth chamber has avolume of 2 milliliters. Uniform mixing can be obtained by pushingfluids through small channels connecting the three growth chambers, eachhaving a volume of 1 milliliter. There is also a 10 microliter reservoirfor sampling located after the growth chamber. The sampling can beperformed via peristaltic pumping of 10 microliter plugs. Besides theconnection to the growth chamber, the sample reservoir is also connectedvia a channel to the sterile water line and a clean air line. Air can beinjected through the sample reservoir to eject any remaining sample intothe sampling container (e.g. an Eppendorf tube), and water can beinjected after that to clean the sample reservoir and remove any cellculture or cells remaining. Clean air can then be sent through thereservoir to dry the chambers so that there would no water left todilute the next sample. This process can be repeated after each samplingstep.

The connections from the RECA micro-bioreactor to the gas manifold areshown in FIG. 6 . All reservoir input valves can share the same gas linesince it is unnecessary to individually control each input valve. Thereservoir pressure can be set to be 1.5 psi (1.03×10⁵ Pa), which islower than that of the mixing pressure of 3 psi (2.06×10⁵ Pa). Thereservoir pressure can be used to ensure that the input to theperistaltic pumps sees the same pressure and is unaffected by externalhydrostatic pressure to ensure consistent pumping volume. The output ofthe reservoir, i.e. the injection valves, can be individually controlledby separate gas lines because these are the valves that determine whichfeed lines are being injected into the growth chamber. Next are the gaslines that control the peristaltic pumps. The mixers can have a separateinput and output line in order to allow flushing of water condensationon the mixer lines, since the air coming into the mixer can behumidified to reduce evaporation of the growth culture. The growthchambers of the micro-bioreactor have large surface to volume ratios andhence, the evaporation rates are generally larger than that for largerbioreactors. Moreover, all three mixer gas lines can be designed to havethe same resistance, to ensure an even mixing rate in the 3 growthchambers. The mixer gas lines can be made wider than the rest of thelines because the air is humidified, and any condensation might clog thelines if the resistance is too high. The last air lines control thevalves to the sampling port. The sampling port consists of a 10microliter sample reservoir and valves to control sampling and automatedcleaning of the sampling port. The holes in the top left corner can besealed with a polycarbonate cover and taped with double sided tape. Theair lines can be connected through a group of 20 barbs located on theleft bottom corner of the chip to the gas manifold.

A gas manifold can be used to connect the solenoid valves to the airlines of the micro-bioreactor. The design of the gas manifold is shownin FIG. 7 . The manifold in this example has 3 layers. The barbconnectors to the micro-bioreactor are situated in the center of the toplayer of the manifold. The middle layer routes the output of thesolenoid valves to the barb connectors that connects the manifold to themicro-bioreactor. The bottom layer routes the main air lines to theinputs of the solenoid valves. Tables 1A-C lists all the valves withtheir numbers as shown in FIG. 7 and the gas connections for easierreferencing.

TABLE 1A for Valves 1-8 Valve Name NO NC 1 Gas Mix 1 Gas Mix 2 (3 Psi)Gas Mix 2 (3 Psi) 2 Reservoir Input Valve On (15 Psi) Valve Off (Atm) 3Injection 1 Valve On (15 Psi) Valve Off (Atm) 4 Injection 2 Valve On (15Psi) Valve Off (Atm) 5 Injection 3 Valve On (15 Psi) Valve Off (Atm) 6Injection 4 Valve On (15 Psi) Valve Off (Atm) 7 Injection 5 (water)Valve On (15 Psi) Valve Off (Atm) 8 Pump 1 Valve On (15 Psi) Valve Off(Atm)

TABLE 1B for Valves 9-16 Valve Name NO NC 9 Gas Mix 2 Nitrogen (3 Psi)Oxygen (3 Psi) 10 Pump 2 Valve Off (Atm) Valve On (15 Psi) 11 Pump 3Valve On (15 Psi) Valve Off (Atm) 12 Sample Reservoir Valve On (15 Psi)Valve Off (Atm) 13 Sample In Valve On (15 Psi) Valve Off (Atm) 14 SampleOut Valve On (15 Psi) Valve Off (Atm) 15 Sample Air In Valve On (15 Psi)Valve Off (Atm) 16 Gas Mix 3 Nitrogen (3 Psi) CO₂ (3 Psi)

TABLE 1C for Valves 17-24 Valve Name NO NC 17 Mixer Bottom Out Mixer Off(Atm) Blocked 18 Mixer Bottom In Blocked Mixer On (3 Psi) 19 Mixer LeftOut Mixer Off (Atm) Blocked 20 Mixer Left In Blocked Mixer On (3 Psi) 21Mixer Top Out Mixer Off (Atm) Blocked 22 Mixer Top In Blocked Mixer On(3 Psi) 23 Reservoir Pressure Res. Off (Atm) Res. On (1.5 Psi) 24 GasMix 4 Available AvailableIn Tables 1A-1C, NO stands for Normally Open and NC stands for NormallyClosed. The selection of which gas lines is normally open or normallyclosed can be selected to be the most common state of the valve, so thatmore often than not, the valve is inactive, to save energy consumption.In particular, Valve 10 (Pump 2) can be set to ‘off’ normally while allthe rest of the valves are set to ‘on’ normally. There are also 4 gasmixer solenoid valves besides the solenoid valves needed for mixing andvalving on the micro-bioreactor. Control of carbon dioxide (CO₂) gasconcentration vs. nitrogen (N₂) gas can be achieved by changing the dutycycle of Gas Mix 3 solenoid valve. Oxygen (O₂) gas concentration can becontrolled via Gas Mix 2 via the same strategy. Then the two outputs canbe mixed together in a 50-50 duty cycle using Gas Mix 1. Gas Mix 4 isavailable for use if any extra valving is needed.

The complete setup is shown in FIG. 8 . A laptop can be used to controla Field-programmable Gate Array (FPGA) board, which can control thesolenoid boards, the heater board, and photo-detector board. Air linescan be connected to a pressure regulator before being connected to thegas manifold. From the gas manifold, the valve lines can be connecteddirectly to the micro-bioreactor. The mixer in lines are connected firstthrough an air resistance line, followed by a 45° C. local humidifierbefore reaching the micro-bioreactor. The mixer out lines from themicro-bioreactor are connected to the water trap, then to the airresistance lines and then only to the gas manifold.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of ex ample only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed is:
 1. A reactor system, comprising: a reactor chamber,comprising: a liquid sub-chamber configured to contain a liquid growthmedium including at least one cell; a gas sub-chamber configured tocontain a gaseous headspace above the liquid growth medium; and agas-permeable flexible membrane separating the liquid sub-chamber fromthe gas sub-chamber; a gas inlet conduit configured to transport gasinto the gas sub-chamber through a reactor chamber gas inlet; a gasoutlet conduit configured to transport gas out of the gas sub-chamberthrough a reactor chamber gas outlet; and a gas bypass conduit externalto the reactor chamber, connecting the gas inlet conduit to the gasoutlet conduit.
 2. The reactor system of claim 1, further comprising ahumidifier configured to humidify the gas transported through thereactor chamber gas inlet.
 3. The reactor system of claim 2, furthercomprising an inlet flow control mechanism configured to regulate theflow of gas through the gas inlet conduit at a rate of equal to or lessthan about 1 milliliter per second.
 4. The reactor system of claim 3,wherein the humidifier is positioned between the inlet flow controlmechanism and the reactor chamber gas inlet.
 5. The reactor system ofclaim 1, further comprising a liquid trap configured to remove liquidvapor from the gas within the gas outlet conduit.
 6. The reactor systemof claim 5, further comprising an outlet flow control mechanismconfigured to regulate the flow of gas through the gas outlet conduit ata rate of equal to or less than about 1 milliliter per second.
 7. Thereactor system of claim 6, wherein the liquid trap is positioned betweenthe flow control mechanism and the reactor chamber gas outlet.
 8. Thereactor chamber of claim 1, wherein the reactor chamber has a volume ofless than or equal to about 50 milliliters.
 9. The reactor system ofclaim 1, wherein the gas-permeable flexible membrane is permeable tocarbon dioxide and/or oxygen gas.
 10. The reactor system of claim 1,wherein the reactor chamber is configured to contain a volume of theliquid growth medium that is equal to or less than about 50 millilitersand equal to or greater than 10 microliters.
 11. The reactor system ofclaim 1, wherein the cell is a eukaryotic cell.
 12. The reactor systemof claim 11, wherein the eukaryotic cell is an animal cell.
 13. Thereactor system of claim 12, wherein the animal cell is a mammalian cell.14. The reactor system of claim 13, wherein the mammalian cell is aChinese hamster ovary (CHO) cell.
 15. A method of operation a reactorsystem, comprising: providing a reactor chamber comprising: a liquidsub-chamber containing a liquid and at least one biological cell, a gassub-chamber configured to contain a headspace above the liquid, whereinthe reactor chamber has a volume of less than or equal to about 50milliliters; a reactor chamber gas outlet conduit configured totransport gas out of the reactor chamber through a reactor chamber gasoutlet; a flexible membrane separating the liquid sub-chamber from thegas sub-chamber; transporting a gas from a gas source through a reactorchamber gas inlet conduit to transport gas into the gas sub-chamber todeform the flexible membrane such that liquid is at least partiallyremoved from the liquid sub-chamber; reducing the supply of the gas tothe gas sub-chamber such that the flexible membrane returns toward itsoriginal position; transporting gas from the gas source through a gasbypass connected to the gas inlet conduit and external to the reactorchamber to remove liquid from the gas inlet conduit; and supplying gasfrom the gas source to the gas sub-chamber at least a second time todeform the flexible membrane such that liquid is at least partiallyevacuated from the liquid sub-chamber.
 16. The method of claim 15,wherein the at least one biological cell is a mammalian cell.
 17. Themethod of claim 16, wherein the mammalian cell is selected from thegroup consisting of primate cells, bovine cells, horse cells, porcinecells, goat cells, dog cells, cat cells, human cells, and hamster cells.18. The method of claim 16, wherein the mammalian cell is a Chinesehamster ovary (CHO) cell.
 19. The method of claim 16, wherein the atleast one biological cell is a cardiac cell, a fibroblast, akeratinocyte, a hepatocyte, a chondrocyte, a neural cell, a osteocyte, amuscle cell, a blood cell, an endothelial cell, an immune cell, or astem cell.